Biochim Biophys Acta Proteins Proteom
September 2024
The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel.
View Article and Find Full Text PDFQβ phage replicase has been the first RNA-directed RNA polymerase purified to homogeneity and intensively studied in vitro. In the mid-sixties, papers on Qβ and related replicases appeared in nearly every issue of the PNAS journal. By 1968, the mechanism of its action seemed to be almost completely understood.
View Article and Find Full Text PDFThe paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion.
View Article and Find Full Text PDFS1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qβ replicase, the RNA-directed RNA polymerase of bacteriophage Qβ. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events.
View Article and Find Full Text PDFQbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs in vitro. Previous studies have shown that Qbeta replicase can initiate and elongate on a variety of RNAs; however, only a minute fraction of them are recognized as 'legitimate' templates. Guanosine 5'-triphosphate (GTP)-dependent initiation on a legitimate template generates a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid, a powerful inhibitor of RNA-protein interactions.
View Article and Find Full Text PDFAt the optimal temperature (65 degrees C), Thermus thermophilus polynucleotide phosphorylase (Tth PNPase), produced in Escherichia coli cells and isolated to functional homogeneity, completely destroys RNAs that possess even a very stable intramolecular secondary structure, but leaves intact RNAs whose 3' end is protected by chemical modification or by hybridization with a complementary oligonucleotide. This allows individual RNAs to be isolated from heterogeneous populations by degrading unprotected species. If oligonucleotide is hybridized to an internal RNA segment, the Tth PNPase stalls eight nucleotides downstream of that segment.
View Article and Find Full Text PDFAn earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers.
View Article and Find Full Text PDFQbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs (RQ RNAs) in vitro. Here we characterize template properties of the 5' and 3' fragments obtained by cleaving one of such RNAs at an internal site. We unexpectedly found that, besides the 3' fragment, Qbeta replicase can copy the 5' fragment and a number of its variants, although they lack the initiator region of RQ RNA.
View Article and Find Full Text PDFWhen PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. Furthermore, because of the spatial separation of the products in the gel, no interference is observedfrom another simultaneously amplified target even if it is present at a 106 higher amount orfrom human nucleic acids that outweigh the target by up to a factor of 1,012, which is often true of clinical samples.
View Article and Find Full Text PDFThe ability of RNAs to spontaneously rearrange their sequences under physiological conditions is demonstrated using the molecular colony technique, which allows single RNA molecules to be detected provided that they are amplifiable by the replicase of bacteriophage Qbeta. The rearrangements are Mg2+-dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10(-9) h(-1) per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of Qbeta replicase, and have a number of biologically important implications.
View Article and Find Full Text PDFExtensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.
View Article and Find Full Text PDFExpression of dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CAT) mRNAs in cell-free Escherichia coli translation systems is greatly enhanced as a result of their insertion into RQ135 RNA, a naturally occurring satellite of phage Q beta. The enhancement is due to protection of the recombinant mRNAs against endogenous ribonucleases and to an increased initial rate of translation in the case of the RQ-CAT mRNA.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
October 1993
Combination of the Q beta replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recombinant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRNA sequence inserted into RQ135(-1) RNA, an efficient naturally occurring Q beta replicase template. The enhancement is associated with a replication asymmetry previously described for the replication of Q beta phage RNA in vivo; the sense (+)-strands are produced in large excess over the antisense (-)-strands. This, in turn, results in increased synthesis of the functionally active DHFR.
View Article and Find Full Text PDFA very efficient replicase template has been isolated from the products of spontaneous RNA synthesis in an in vitro Q beta replicase reaction that was incubated in the absence of added RNA. This template was named RQ135 RNA because it is 135 nucleotides in length. Its sequence consists entirely of segments that are homologous to ribosomal 23 S RNA and the phage lambda origin of replication.
View Article and Find Full Text PDFThe analysis of the deletion derivative of pBS359 obtained as a result of sodium bisulphite mutagenesis and of recombinant derivatives pBS361-pBS363 permitted to map genes of the broad-host-range pBS222 plasmid which participate in replication, maintenance and mobilization. These genes are localized within the coordinates 0.2 to 2.
View Article and Find Full Text PDFMol Gen Mikrobiol Virusol
August 1985
The technique of localized in vitro mutagenesis in the cohesive ends of plasmid pBR322 DNA has been elaborated (separately for BamHI and HindIII sites). Plasmid DNA digested by restriction endonucleases has been treated with sodium bisulphite deaminating cytosine to form uracil in single stranded DNA (cohesive ends of the plasmid). The mutagenized plasmid DNA, free of mutagen, has been treated with bacteriophage T4 ligase.
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